1. Field of the Invention
This invention relates to the characterization of the role that a toll-like receptor (TLR) motif in the amino acid sequence of the non-structural protein NS4B of highly virulent Classical Swine Fever Virus (CSFV) strain Brescia plays during infection in the natural host and to the utilization of a strategy for altering CSFV virulence by replacing the TLR motif with a different amino acid triplet to obtain an attenuated recombinant virus, NS4B-VGIv, which protects swine from challenge with virulent BICv.
2. Description of the Relevant Art
Classical swine fever (CSF) is a highly contagious disease of swine. The etiological agent, CSF virus (CSFV), is a small, enveloped virus with a positive, single-stranded RNA genome and, along with Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), is classified as a member of the genus Pestivirus within the family Flaviridae (Becher et al. 2003. Virology 311: 96-104). The 12.5 kb CSFV genome contains a single open reading frame that encodes a 3898-amino-acid polyprotein and ultimately yields 11 to 12 final cleavage products (NH2-Npro-C-Erns-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH) through co- and post-translational processing of the polyprotein by cellular and viral proteases (Rice, C. M. 1996. In: Fundamental Virology, 3rd edition, Knipe et al., eds., Lippincott Raven, Philadelphia, Pa., pages 931-959).
The non-structural glycoprotein 4B (NS4B) of CSFV is one of the non-structural proteins of classical swine fever virus. NS4B has previously been poorly characterized in terms of its functionality. Using genomic analyses we have found that NS4B possesses a specific motif that is found in proteins of the Toll-Like Receptor (TLR) family. In general, TLRs are pattern-recognition receptors that recognize structurally conserved molecules derived from microbes. Their expression is readily modulated in response to pathogens and plays an important role in mounting the host immune response. Although the function of natural ligands of swine TLRs is still under study, these molecules are highly conserved within the Vertebrata, suggesting activities similar to other published vertebrate TLRs and their ligands.
Strategies for controlling disease in the event of a CSFV outbreak include the production of rationally designed live attenuated vaccine CSFV strains. Here, we report the effects of modification of the TLR of NS4B on viral infectivity and virulence in swine.